I. Clinical Studies of Uveal Coloboma Since initiating this research, I have recruited and examined over 100 famlies where at least one member is affected by uveal coloboma. All probands and their first degree relatives (when available) have complete ophthalmic exams. General physical examinations and targeted systemic testing (e.g., renal ultrasounds, echocardiograms) were performed on probands, as needed. Lymphoblastoid cell lines were established on all participants for candidate gene analysis. Preliminary analysis of these patients has resulted in four important clinical observations. First, patients with coloboma often have thickened corneas as part of their developmental defect. This affects the measurement of their eye pressures. Second, I have identified a new syndrome in which missing thoracic and/or lumbar vertebrae co-segregates with coloboma. This was seen in two, unrelated autosomal dominant pedigrees. We have recently published this finding as part of a broader paper on systemic findings in patients with uveal coloboma. We are currently pursuing a whole-exome strategy to try to find the gene responsible. Third, I have determined the yield of systemic testing on patients with apparent isolated coloboma, resulting in the publication in the American Journal of Ophthalmology mentioned above.. Fourth, I have defined microforms of coloboma that aid in genetic counseling of families. II. Laboratory Studies of Uveal Coloboma A. Mouse Models of Coloboma. 1. A novel Pax2 mutant mouse model of coloboma. My lab identified and characterized a mouse model of autosomal dominant congenital optic nerve excavation caused by a missense mutation predicted to change a highly-conserved threonine to alanine in the paired domain of the Pax2 gene. Pax2 is dynamically expressed at the closing edges of the optic fissure and homozygous mutation results in uveal coloboma. Details of our characterization of this model and the Pax2 mutation have been published in PLoS Genetics. More recently, we have used this model system to look at the effects of Pax2 loss-of-function on other genes of interest, such as Nlz1 and Fat1/Fat4. 2. The RICO Mouse Model of Coloboma The RICO mouse arose from the random insertion of a transgene (NSE-VEGF) on chromosome 13 of C57BL/6 mice. Although the transgene is not expressed in the adult or embryonic mouse, its insertion led to a classic coloboma phenotype. The region of insertion did not suggest any previously-identified coloboma genes. This phenotype is dominant and does not significantly affect the viability or fertility of heteorygous mice. We have recently determined that homozygous mice are rarely viable postnatally. Tiling arrays of chromosome 13 show no evidence of major deletion. Pax2 expression is not affected in RICO embryos, indicating that the responsible gene is either independent or downstream of Pax2. We pursued a high-throughput sequencing approach to identify the transgene insertion and have identified both ends of a junctional fragment. This region of mouse chromosome 13 is a gene desert, but is well-conserved across species, suggesting that it contains an important regulatory motif. We are currently using FISH with several BAC clones in the region of the insertion--which appears to have caused an inversion in the process. B. Identification of coloboma candidate genes by molecular characterization of gene expression during optic fissure closure. 1. Zfp503 and Zfp703 Our previous work, published in PNAS, identified two zinc-finger motif-containing genes, Zfp703 and Zfp503 to be important in regulating optic fissure closure in zebrafish. We have created knockout mice for both Zfp703 and Zfp503 and documented germline transmission. We have demonstrated that homozygous knockout of Zfp503 is lethal in late embryonic development. The reason for non-viability is currently being pursued. We have also demonstrated that homozygous embryos develop coloboma, confirming our results from zebrafish work in a mammalian system. In addition, we have characterized the insertion site in the Zfp703 mouse, confirming its homologous recombination. We have determined that homozygotes are not viable postnatally, although the reason is currently unclear. We are currently examining the eye phenotype of these mice. In addition, we have made a more careful study of the zfp703 zebrafish morphant phenotype. We have identified that morphant fish have several important phenotypes such as cystic kidneys and abnormalities in heart development. As such, this model likely represents a syndromic form of coloboma. We have extended our mutation screen to include patients with syndromic forms of coloboma in hopes of identifying causative mutations. Current results are ambiguous are being confirmed using independent methods. In addition, we have identified that zfp703 morphants show a reduced rate of cell division in the optic cup, suggesting that the mechanism of coloboma is non-apposition of the edges of the fissure. 2. FAT protocadherins Another gene family that was suggested by our laser-capture screen was the FAT protocadherins. As previously described, we have found that Fat1 and Fat4 are the members of this family that are most highly-expressed during embryonic eye development and that homozygous knockout of Fat1--but not Fat4--results in coloboma. We have shown that the coloboma in Fat1-/- mice is not the result of a global patterning defect and that the eyes of these embryos are approximately normal size until the time of optic fissure closure. The rate of cell division in the developing optic cup is mildly elevated compared to wild-type and there is no obvious change in the rate of cell death. Real-time PCR of embryos on a panel of genes has revealed RPE-specific changes in several important cell adhesion molecules, suggesting that that mechanism of non-closure is directly related to non-adhesion. We are currently organizing our data for publication. 3. 5q deletion and coloboma We have identified a family with an interstitial deletion on chromosome 5q and coloboma. High throughput sequencing of the proband and his parents did not reveal any mutations in known coloboma genes, providing additional support that the chromosomal deletion and the phenotype were related. Previous work identified the aldehyde-dehydrogenase 7, which is highly expressed at the edges of the closing optic fissure in zebrafish, as a potential candidate. Morpholino studies show a coloboma, facial bone malformations and fin shortening phenotype with ALDH7 knockdown, similar to the patient phenotype. Unlike Fat1, knockdown of aldh7a1 results in reduced cell division in the optic cup. The mechanism of coloboma is therefore most likely a failure of apposition of the edges of the fissure at the right time in development. This effect can be partially rescued with nlz1 mRNA and with folic acid, suggesting that these factors are functionally downstream of aldh7a1. We are currently preparing a manuscript to publish these results.